Microemulsion of nutrient substances

ABSTRACT

There is disclosed a nutrient composition to be added to a hydrophobic medium for culturing microorganisms comprising a micro-emulsion of an aqueous solution of nutrient material in a liquid immiscible with water. As the source of nitrogen, compounds soluble in water are used, such as urea or ammonium nitrate, sulphate or phosphate, and, as the source of phosphorus, compounds soluble in water are used. The medium sould also contain a directly-assimilable source of carbon. Application of this composition for culturing of microorganisms allows operations to be carried out which comprise the degrading of hydrocabons covering an area of water or ground; an application of particular interest for operation is for combatting oil pollution of the sea by biological degradation. As the source of nitrogen, compounds soluble in water are used, such as urea or ammonium nitrate, sulphate or phosphate, and, as the source of phosphorus, compounds soluble in water are used. The medium should also contain a directly-assimilable source of carbon. Application of this composition for culturing of microorganisms allows operations to be carried out which comprise the degrading of hydrocarbons covering an area of water or ground; an application of particular interest for operations is for combatting oil pollution of the sea by biological degradation.

This is a division of application Ser. No. 302,961 filed Sept. 17, 1981now U.S. Pat. No. 4,401,762.

The present invention relates to a novel type of microemulsion, namelythe thermodynamically-stable microemulsion of an aqueous solution ofnutrient materials, the external phase of which is constituted by ahydrophobic medium. It comprises a process of production of such amicroemulsion and applications of it in areas of utilization ofmicroorganisms, particularly in the culture of microorganisms in ahydrophobic medium.

Industrial operations making use of cultures of various microorganisms,particularly bacteria and fungi, are of particular interest at thepresent time. Numerous food industries, manufactures of medicaments,purifications etc. are based upon such operations. Most frequently, theoperation comprises two stages: Firstly, culture of the microorganismconcerned takes place in an appropriate nutrient medium to achievegrowth of a sufficiently numerous population; in the second stage, thispopulation is placed in contact with materials which it is desired tosubject to the action of the microorganisms. Operations in vessels donot involve special difficulties from the standpoint of, preliminaryculture, that is, multiplication, being effected--depending upon thecase--in a separate enclosure or in the same vessel where the secondoperative phase takes place. In contrast, when the operations arecarried out in nature, on large areas of ground or water, as is the casefor example in the elimination of hydrocarbon slicks by microbiologicaldegradation at sea, on beaches, in flowing water or on lakes, thepreliminary multiplication of the microorganisms used presentsdifficulties. In practice, it is necessary to provide the culture withnutrient materials, that is sources of C, N and P, as well as traceelements, to ensure growth of the organisms in question. When thestandard sources, such as hydrides of carbon, nitrates or ammonicalsalts and phosphates are soluble in water, they cannot remain in theextended superficial layer to be treated, where a strong growth of themicroorganisms used should occur. These substances diffuse into thewater or the adjacent ground and are thus under-used by the culture.

To remedy this, various methods have been employed up to the present.One of them consists in enrobing in paraffin granules of the nitrogenouscompound and solid phosphates in order to make them available to themicroorganisms in this form, as indicated in U.S. Patent SpecificationNo. 1,959,127. In a variant, according to the U.S. Patent SpecificationNo. 3,883,397, the lipophilic enrobing compound is a fatty acid salt inplace of paraffin. However, these methods do not allow themicroorganisms to have the desired nutrient rapidly available. The fattyenrobing material is difficult to penetrate, that is, to degrade, in theabsence of external nitrogen and phosphorus. Also, the action ofbacteria or fungi itself is slow and requires weeks or months. Anotherproposed solution consists in utilizing, as the sources of N and P,compounds insoluble in water but soluble in hydrocarbons, particularlyphospho-amino-lipids, as described in French Published Specification No.2,172,796. However, nitrogenous compounds soluble in oil generally havea very low nitrogen content and, under these circumstances, thebiological degradation of petroleum hydrocarbons in the sea requires twoto three months. In French Published Specification No. 2,230,401, theuse has also been indicated of amides, organic ammonium salts andphospho-amino-lipids in solution in a petroleum solvent, the solutionbeing emulsified in water. The emulsion obtained is atomized over ahydrocarbon oil slick floating on the water for biodegradation of theslick. This method requires large proportions of the aqueous emulsionand the result is only obtained after several weeks.

The present invention provides a novel solution to the supply ofnutrient substances, soluble in water, to a hydrophobic organic layer.Whether this layer--which can be a hydrocarbon--floats on water or isdisposed on the ground or on a support comprising a constructionalmaterial, the nutrient substances provided according to the inventionremain principally in the hydrophobic layer and permit rapidmultiplication of the microorganisms if sources of them are present.

The novel process according to the invention consists in making amicroemulsion of the water-in-oil type, the internal phase of which isan aqueous solution of nutrient materials and the external phase ofwhich is a liquid immiscible with water, and adding this microemulsionto the hydrophobic layer which is to be degraded. The microemulsion cancontain sources of the appropriate microorganisms, if the medium to betreated contains none or does not contain sufficient amounts.

It will be understood, as is known in the art, that the microemulsioncontains at least one surface-active agent and an auxiliary agent, whichhave served in its preparation.

Thus, contrary to the prior technique, the nutrient substances areneither utilized in the solid state nor in solution in a solventimmiscible with water nor in an aqueous macroemulsion, but in the formof a microemulsified aqueous solution in a liquid miscible with thehydrophobic layer to be biodegraded, that is, in the form ofmicrodispersed so-called inverse droplets, the diameter of which rangesfrom 80 to 600 Ångstroms and in particular from 100 to 200 Ångstroms.This unexpected form leads to the remarkable result that thebiodegradation can be realized in several days, in place of the weeks ormonths required by known processes.

As regards the source of nitrogen, in the microemulsions according tothe invention, various compounds soluble in water and assimilable by themicroorganisms can be employed. These are, for example, ammoniumnitrate, sulphate and/or phosphae, urea, proteins, peptones, etc. Asurea is the source richest in nitrogen and is very soluble in water, itis particularly appropriate because it allows highly concentratedaqueous solutions to be produced. For example, solutions of ureacontaining 10% to 60% by weight can be employed, that is, 11 to 150parts of urea per 100 parts of water.

Phosphorus can also be provided in the solution in one of the customaryforms, namely alkali metal or ammonium phosphates or phosphites.According to a particular embodiment of the invention, phosphorus isprovided in the form of a surfactant compound, such as a higher alkylphosphate or a lecithin, for example. The source of phosphorus and thesurface-active agent of the microemulsion are thus in the same molecule.

In various industrial operations comprising the culture ofmicroorganisms, it is necessary for the pH of the medium to be adjustedto the value most favourable to bacterial growth. In general, this pHshould be in the vicinity of neutrality and it is possible to add eitherphosphoric acid as a source of phosphorus, if the medium has to beacidified, or ammonia as a source of nitrogen, if the medium has to beneutralized by a base.

In the case of bacterial degradation of hydrocarbons by themicroorganisms cited below, the need for phosphorus is much less thanfor nitrogen; expressed in weight, the P/N ratio can range from 0.02 to0.2 and preferably from 0.05 to 0.15. From the standpoint of growth, themost favourable P/N ratios are those as near as possible to 0.05.

When the liquid immiscible with water and preferably lipomiscible,forming the external phase of the microemulsion, or the hydrophobiclayer to be degraded is utilizable by the microorganisms as the sourceof carbon, it is no longer necessary to add other assimilable carboncompounds to the microemulsion. However, if the external phase and thelayer to be degraded are attacked with difficulty by the microorganisms,at least at the beginning, it is useful to include in the nutrientsolution a readily-utilizable carbon source, for instance solublehydrides of carbon, thus allowing rapid onset of multiplication of themicroorganisms.

As in all cultures, trace elements are necessary, particularly salts ofFe, Mg, K etc and, a very small dose can be added to the nutrientsolution, in known manner.

It can be seen that, in order to obtain a microemulsion according to theinvention, a surface-active compound capable of producing one isnecessary. The choice of a suitable compound, by a person skilled in theart, can be made from various groups of nontoxic surface-active agentsfor the microorganisms present. For example, fatty alcohol sulphates,sulphosuccinates, oxyethylenic sorbitan esters, oxyethylenic alcohols,acids or oils, esters of saccharose, amino-acids, alpha-amido-aminoacids, taurines, sarcosines, polyglycols, higher alkyl phosphates etc.can be used. This list is not limitative and other surfactants can beutilized, in particular those which have dispersant properties vis-a-vishydrocarbons.

Preferably, the hydrophilic-lipophilic balance of the emulsifying agentsemployed is from 10 to 17 and most preferably from 11 to 15.

When operations are concerned which are carried out outside, thesurfactant itself must be biodegradable, in order to avoid environmentalpollution.

As with the surfactants, the choice is equally vast as regards theco-surfactant necessary for the formation of the microemulsion. Suchco-agents are well-known in the art and thus they do not need to belisted here. It can merely be noted, in a non-limitative way, that it ispossible to use nitrogenous compounds, such as carbamates, amides oramine salts. The viscosity of the microemulsion can be considerablyreduced by the addition of an alcohol, particularly C₆ to C₁₂, an etheror an ester of a polyol, particularly glycol. This considerablyfacilitates manipulative operations.

When the external phase of the microemulsion must be miscible with thehydrophobic liquid to be biodegraded, it is necessarily chosen accordingto the nature of this liquid. In the most important practical case,where the latter is constituted by petroleum hydrocarbons, thelipomiscible external phase can be constituted for example by aliphatic,aromatic or naphthenic hydrocarbons or by so-called mineral oils, thatis mixtures of such hydrocarbons. This type of external phase isattacked with difficulty by bacteria, when these have not undergonesufficient adaptation. It is thus preferable to utilize vegetable oranimal oils, which can serve as the source of carbon because they areutilizable by microorganisms. These oils or preferably theircorresponding fatty acids permit rapid development of the microorganismsnecessary for the degradation of the hydrophobic layer, particularlycrude petroleum.

The weight ratio between the lipomiscible liquid, that is the externalphase of the microemulsion, and the aqueous solution to be emulsifiedshould generally be greater than 0.2 This ratio is selected so that theaqueous solution forms the internal phase. The choice of surfactants andco-surfactants is effected according to the nature of the lipomiscibleliquid and depending upon the concentration of the salts dissolved inthe aqueous phase. The basis for this are the concepts of formulation ofmicroorganisms, known per se.

The process of the invention is applicable to a large number ofmicroorganisms and in particular to those which allow the degradation ofhydrocarbons. Thus, the invention can be applied to the utilization ofbacteria such as Pseudomonas, Acinetobacter, Flavobacterium,Artrobacter, Corynebacterium etc. The microorganisms can also be fungi.

While the invention is of great interest for various operations ofbiodegradation effected outdoors, it can also be of use in variousmanufacturing operations in vessels, whenever a hydrophobic layer of asubstance is employed in the process. For example, it appliesadvantageously in the manufacture of proteins from hydrocarbons bydegradation of the latter with the aid of bacteria and/or fungi. In allcases, the remarkable dispersion of aqueous nutrient substances withinthe hydrophobic phase, obtained through the invention, leads to a veryrapid multiplication of the microorganisms. An appreciable gain in timein these operations thus results.

Among applications in the open, water or ground areas, the mostimportant is the degradation of hydrocarbons distributed accidentally.For the reason explained above, that is the fact that the solublenutrient substances remain in the layer treated instead of beingentrained by the water, the invention has a considerable value forcombatting marine pollution. However, the same principle applies tooperations such as the cleansing from banks, reservoirs, ground areas,containers etc. of hydrocarbon deposits which pollute them. Otherapplications comprise the distribution of nutrients on agriculturalcultures.

Microorganisms are generally present in the medium being treated.However, it is sometimes necessary to effect seeding, when the initialpopulation is judged to be too low or if the medium contains noappropriate bacteria.

In a particular embodiment of the present invention, urea is utilized asthe nitrogenous nutrient substance. It has been found that this compoundalso plays the part of the co-surfactant and it is thus no longernecessary to add another co-surfactant. On the other hand, as phosphoruscan advantageously be provided by alkyl esters of phosphoric acid, whichprovide surfactant properties, the composition of the nutrient solutionis simplified by the fact that it is possible to employ urea and thephosphoric ester without any other additive. It is neverthelessrecommendable to add liquids permitting reduction of the viscosity ofthe microemulsion. Various examples of such additives have been citedabove. In a particular embodiment of the invention, the butyl ether ofethylene glycol has given excellent results.

The lipomiscible liquid particularly suitable for the external phase ofthe microemulsion, according to the invention, can be constituted by oneor more esters of fatty acids, such as lauric, myristic, palmitic,arachidic, oleic, stearic, caproic and caprylic acids etc. Glycerides ofsuch acids constitute very readily available industrial products,because they are vegetable and animal oils. Thus, oils such asarachidic, whale, rape, linseed, maize, olive, sesame and tall oils etc,can be used. Fatty acids themselves are particularly suitable, ifrequired in appropriate mixtures, in order to remain liquid at ambienttemperatures. Thus fatty acids are useful, particularly those containingC₆ to C₁₈, such as caproic, oenanthylic, caprylic, lauric, palmitic,oleic, lineoleic or stearic for example. To those fatty materials whichare not liquids at ordinary temperatures, it is suitable to addhydrocarbons, for example petroleum or gas oil in the proportion ofabout 5% to 50%. Fatty alcohols, that is from C₆ to C₂₄, are equallysuitable.

In a particular case, where the aqueous solution contains urea andlauryl and/or oleyl phosphates marketed for example by Hoechst under thename "Hostaphat", the preferred nitrogen content of the entiremicroemulsion is about 4% to 10% by weight or most preferably 5% to 8%.The weight ratio of nitrogen in the lipomiscible liquid is generallyfrom 0.1 to 0.4 and preferably 0.15 to 0.35.

In a general manner, the preferred microemulsions according to theinvention comprise by weight 10% to 30% of water, 4% to 10% toassimilable nitrogen in the form of nitrogenous compounds, 5% to 35% ofa C₁₀ to C₁₈ alkyl phosphate or an ethoxylated phenol alkyl phosphate,0% to 20% of an alkyl ether of an alkylene glycol and 20% to 50% of analiphatic ester, acid and/or alcohol. The last compounds can compriseliquid hydrocarbons, such as petroleum or its derivatives, for examplein the ratio of 5% to 90%.

A variant of the invention comprises an improvement which allows a veryrapid action of the microorganisms to be obtained. This renders possiblethe degradation of hydrocarbons in a very short time, by utilizing forthis purpose a very large number of microbes. It has been discoveredthat, while the best nutrient substance is urea, a part of themicroorganisms normally present in seawater and capable of degradinghydrocarbons do not develop and consequently do not participate in thedesired degradation. According to the present variant, this fraction ofthe microorganisms which remain "inactive" can be caused to develop andparticipate in the degradation of the hydrocarbons, if the nitrogenousnutrient material is accompanied by one or more nitrogenous materials ofa chemical composition substantially different from the first.Particularly favourable results are obtained when the first nutrientmaterial is urea and the second is constituted by one or moreamino-acids.

It follows that a microemulsion according to the invention intended forthe microbiological treatment of a hydrocarbon material preferablycontains an aqueous solution of at least two nitrogenous compounds whichare substantially different from the chemical standpoint. Thus, forexample, if the first nutrient substance is a salt such as ammoniumsulphate, phosphate or nitrate, the second is constituted by an amine,an amide, a protein, an amino-acid or another non-ammoniacal compound.

When the nutrient solution comprises urea, the second nitrogenouscompound is, for example, ammonium sulphate, phosphate or nitrate or anamino-lipid and particularly an amino-acid. The relative proportions ofthe two kinds of nitrogenous materials can vary largely, depending uponthe nature of the microbial flora of the medium where the process of theinvention is applied. Most frequently, the effective proportion of urea,expressed as nitrogen, is from 50% to 99% of the total nitrogen, that isthe nitrogen of the amino-acid represents 50% to 1%. In certain aqueousmedia, about 1% to 10% of nitrogen can be sufficient in this secondform, in order to obtain excellent results.

The amino-acids advantageously utilizable according to the presentinvention can be selected from all those which are found in nature andfrom synthetic amino-acids. By way of non-limitative examples, use canbe made of glycine, alanine, serine, cysteine, valine, glutamine,leucine, lysine, arginine, prolie, tyrosine, aspartic and glutamic acidsetc. For reasons of economy, it is useful to utilize the materialsobtained from natural products which generally contain a series ofseveral amino-acids. This is the case for example with wines made fromsugar beet, extracts obtained by the maceration of various plants,particularly maize cobs, yeast extracts, products of the hydrolysis ofproteins, dairy by-products etc.

The invention leads to the unexpected conclusion that, if a nitrogenousnutrient material gives good results alone and if the same applies toanother nitrogenous material of a different chemical nature when usedalone, the degradation of hydrocarbons by microorganisms is much better,if the two materials are utilized conjointly, the total concentration ofassimilable nitrogen being the same.

Thus, microemulsions according to the invention allow a degradation ofmore than 80% of crude petroleum distributed on seawater to be obtained,for example, in seven days, when the nutrient solution contains urea oramino-acids. But the same result is obtained in six days, if the ureaand the amino-acids are present conjointly in the solution, the totalnitrogen concentration thereof being the same as in the two precedingcases.

The invention is illustrated by the series of non-limitative exampleswhich follow.

EXAMPLES 1 TO 11

For each of the tests, a certain volume of a 50% by weight aqueous ureasolution was mixed with a volume of oleic acid in the presence of acertain quantity of surfactants constituted by a mixture of C₁₂ to C₁₈fatty alcohol phosphoric esters marketed by Hoechst Company under thename "Hostaphat". In certain of the tests, the butyl ether of ethyleneglycol was also added to lower the viscosity. The range of temperaturesin which the microemulsion obtained was stable was determined.

Table 1 on the following page indicates the compositions of themicroemulsions so prepared, the stability ranges of the latter and theirviscosity. It can be seen that an excellent stability in the range from0° C. to more than 40° C. can be obtained according to Examples 4, 5, 6,8, 10 and 11. As regards viscosity, it was found that, without theaddition of the butyl ether of ethylene glycol, it is very high(Examples 1 and 2). In contrast, this addition reduces it to veryacceptable values (Examples 3 to 11).

                                      TABLE 1                                     __________________________________________________________________________                       1   2   3   4   5   6   7   8   9   10  11                 __________________________________________________________________________    Water              11.1                                                                              11.7                                                                              13.8                                                                              14.4                                                                              18.3                                                                              20.1                                                                              20.9                                                                              21.9                                                                              19.2                                                                              20.2                                                                              19.2               Urea               11.1                                                                              11.7                                                                              13.8                                                                              14.4                                                                              18.3                                                                              20.1                                                                              20.9                                                                              17.2                                                                              15.8                                                                              16.6                                                                              15.7               Oleic Acid         44.4                                                                              46.7                                                                              36.8                                                                              38.4                                                                              36.6                                                                              32.1                                                                              27.9                                                                              27.9                                                                              28  29.4                                                                              28                 Butyl ether of ethylene glycol                                                                   0   0   10.6                                                                              9.8 10.7                                                                              11.6                                                                              14.4                                                                              14.4                                                                              17.5                                                                              16.2                                                                              10.5               Lauryl Phosphate (KL 340)                                                                        33.3                                                                              --  25  --  --  --  --  --  19.5                                                                              8.8 19.5               Oleyl Phosphate (KO 380)                                                                         --  30  --  23  --  --  --  --  --  --  --                 Ethoxylated Phenol Alkyl Phosphate                                                               --  --  --  --  16.1                                                                              16.1                                                                              15.9                                                                              15.9                                                                              --  8.8 --                 Mono Ammonium Phosphate                                                                          --  --  --  --  --  --  --  2   --  --  --                 % Nitrogen         5.2 5.45                                                                              6.44                                                                              6.72                                                                              --  --  --  --  7.4 --  --                 Nitrogen/Oleic acid ratio                                                                        0.117                                                                             0.117                                                                             0.175                                                                             0.175                                                                             0.23                                                                              0.29                                                                              0.35                                                                              0.31                                                                              0.26                                                                              0.26                                                                              0.26               Phosphorus/Nitrogen ratio                                                                        0.154                                                                             0.064                                                                             0.09                                                                              0.043                                                                             0.099                                                                             0.09                                                                              0.086                                                                             0.063                                                                             0.064                                                                             --  --                 Stability Range °C.                                                                       10-44                                                                             10-46                                                                             1-44                                                                              0-44                                                                              0-40                                                                              0-47                                                                              12-57                                                                             0-40                                                                              0-36                                                                              0-42                                                                              0-45               Viscosity in cps at 20° C.                                                                1200                                                                              2000                                                                              167 265 203 197 141 98  61  --  --                 __________________________________________________________________________

EXAMPLES 12 TO 16

Microemulsions were prepared with 33% lauryl phosphate. The lipomiscibleliquid was oleic acid in a quantity equal to twice that of the aqueoussolution of urea and phosphate. The percentage of urea in the aqueousphase was varied. The results below give the maximum temperature atwhich the microemulsion was still stable.

    ______________________________________                                        % Urea in the aqueous                                                                         Upper limit of the temp-                                      phase           erature °C.                                            ______________________________________                                          22.2          36                                                            25              32                                                            33              38                                                            50              35                                                            56              No microemulsion                                              ______________________________________                                    

The results show that microemulsions viable in practice, stable up toabout 36° C. can be obtained with concentrations of urea ranging up to50%, but not above that.

EXAMPLES 17 TO 19

With a 50% aqueous solution of urea and the addition of oleyl phosphatecontaining 30% of the monoethyl-ether of ethylene glycol, threemicroemulsions were prepared containing varying proportions of oleicacid. The stability ranges of these products were as follows:

    ______________________________________                                        Weight ratio of the                                                                            Surfactant Stability                                         urea oleic acid  (Oleyl phos-                                                                             range                                             solutions        phate)     °C.                                        ______________________________________                                        0.5              25.3       0-65                                              0.625            24.2       0-59                                              0.75             26.7       0(gel)-64                                         ______________________________________                                    

This shows that above an aqueous solution/oleic acid ratio of about 0.65and for a fixed quantity of the mono-ethyl-ether of ethylene glycol of30%, the microemulsion is difficult to utilize because it tends to gel.In contrast, below this ratio, excellent stability is confirmed.

DEGRADATION OF PETROLEUM HYDROCARBONS EXAMPLE 20

30 l of seawater sterilized at 120° C. for 2 hours was introduced into a50-liter fermenter. On the surface of the seawater, 30 ml of 34° APIcrude petroleum containing 75% saturated hydrocarbons and 25% aromaticswas spread out. The petroleum layer had a thickness of 0.5 mm. On thislayer, 6 ml of one of the microemulsions of the foregoing Examples wasatomized. The medium was thus seeded with sources derived from theseawater. These sources were obtained by culturing for 24 hours on anaqueous solution of glucose. They contained Pseudomonas in a major part.

After this inoculation, the sources in the petroleum samples wereassayed; 2.5×10³ to 4×10⁴ microbes per ml were thus formed.

Aerobic culture was then carried out by agitating the contents of thefermenter with an agitator rotating at 400 revs per min, while 120liters of sterilized air per hour were blown in. This aerationcorresponds substantially to that which takes place naturally at sea.

After 40 hours, a further assay was effected. The results are indicatedbelow.

After seven days, the rate of degradation of the petroleum wasdetermined by extraction of the residual hydrocarbons with CCl₄ andinfrared measurement. The results obtained with the microemulsions ofExamples 1, 2, 3, 10 and 11 are indicated below.

    ______________________________________                                                                        Rate of degra-                                Microemulsion                                                                           Initial   Source assay                                                                              dation after                                  of        source    after 48 hours                                                                            7 days                                        ______________________________________                                        Ex.    1      2.5 × 10.sup.3                                                                    2.5 × 10.sup.8                                                                    83%                                                2      2   × 10.sup.4                                                                    9.5 × 10.sup.8                                                                    90%                                                3      1.5 × 10.sup.4                                                                    2.5 × 10.sup.8                                                                    90%                                               10      4.5 × 10.sup.3                                                                    2.5 × 10.sup.8                                                                    86%                                               11      1.5 × 10.sup.3                                                                    7.5 × 10.sup.7                                                                    92%                                         ______________________________________                                    

EXAMPLE 21

A test was carried out according to the mode of operation described inExample 20, but non-sterilized seawater are introduced into thefermenter and the medium was not seeded with supplementary bacteria.

The results are:

    ______________________________________                                                                        Rate of degra-                                Microemulsion                                                                           Initial   Source assay                                                                              dation after                                  of        source    after 40 hours                                                                            7 days                                        ______________________________________                                        No 11     2 × 10.sup.3                                                                      5.5 × 10.sup.7                                                                      82%                                           ______________________________________                                    

EXAMPLE 22

A test was carried out according to the mode of operation of Example 20,but the seawater was replaced by natural water into which variousmineral constituents of seawater had been added, namely 30 ppm of traceelements, particularly iron, magnesium and potassium. 6 ml ofmicroemulsion No. 11 was atomized and the medium was seeded with aculture of bacteria.

After 7 days, the rate of degradation of the petroleum was determined byextraction of the residual hydrocarbons with CCl₄ and infraredmeasurement; the rate was 90%.

EXAMPLES 23 TO 24

Biodegradation tests on crude Arabian petroleum were effected at theedge of the sea in a vessel having a depth of 2 meters, divided intofour compartments each 3 m×3 m in horizontal section. The compartmentscould be isolated or connected together and all were capable ofreceiving seawater.

A hydro-ejector pump system ensured light agitation of the water andrenewal of the air in the vessel.

Into each compartment, 15.6 m³ of seawater and 4 liters of the petroleumindicated above were introduced, thus providing a layer 0.45 mm thick onthe surface of the water.

One of the compartments served as a control; the water and the petroleumcontained in it were agitated as in the other compartments, but receivedno additive. At the end of the test, the losses of petroleum due tonatural causes were determined in order to take them into account inevaluation of the degradation caused by the additions according to theinvention.

In each of the three other compartments, 0.4 l of a microemulsion of anutrient solution was introduced, comprising in % by weight:

    ______________________________________                                        urea             17.0                                                         clear water      20.8                                                         butyl glycol     10.8                                                         lauryl phosphate 21.1                                                         oleic acid       30.3                                                         ______________________________________                                    

At the start of the test, the microflora of the seawater in the tankcomprised 10² bacteria per ml. After 7 days the quantities of petroleumwhich had disappeared from the compartments were determined. The Tablebelow indicates the amounts in % of the initial quantity at twodifferent temperatures.

    ______________________________________                                                 Control                                                                              Example 23                                                                              Control  Example 24                                          12° C.                                                                        12° C.                                                                           18° C.                                                                          18° C.                              ______________________________________                                        % of disap-                                                                              5         5        17.5   17.5                                     pearance due to                                                               natural causes                                                                % due to treat-                                                                          0        58        0      61.3                                     ment according to                                                             the invention                                                                 % total disap-                                                                           5        63        17.5   78.8                                     pearance                                                                      ______________________________________                                    

As can be seen, even at the relatively low temperature of 12° C., thebiodegradation obtained in seven days by the treatment of the inventionis remarkable. In contrast to most of the known processes without theuse of a culture of microorganisms, this was obtained with the sole useof those existing in the seawater.

EXAMPLE 25

30 ml of seawater were introduced into a 50-liter fermenter. On thesurface of this water, there was spread 30 ml of crude 34° APIpetroleum, containing 75% of saturated hydrocarbons and 25% aromatics.On the petroleum layer thus formed, having a thickness of 0.5 mm, 6 mlof the microemulsion having the following weight composition wasatomised:

    ______________________________________                                        urea                 17.3%                                                    water               21.5                                                      butyl ether of ethylene glycol                                                                    10.8                                                      lauryl phosphate    23.7                                                      oleic acid          26.7                                                      (8.07% of nitrogen) 100.0                                                     ______________________________________                                    

Assay of the bacteria in the petroleum indicated the presence of 10²bacteria per ml.

Aerobic culture was then carried out by agitating the contents of thefermenter with an agitator rotating at 400 revs per min while 120 litersof sterilized air per hour were blown in. This aeration correspondedsubstantially to that which takes place naturally at sea.

After 48 hours, a new assay was effected. It showed the presence of2.5×10⁸ bacteria per ml.

After 7 days, the rate of degradation of the petroleum was determined byextraction of the residual hydrocarbons with CCl₄ and infraredmeasurement. This rate was 83%.

EXAMPLE 26

Identical operations to those of Example 25 were effected, but in themicroemulsion a part of the urea was replaced by the amino-acidDL-valine. The weight composition of the microemulsion was:

    ______________________________________                                        urea                 16.8%                                                    DL-valine            2.0                                                      water               20.5                                                      butyl ether of ethyleneglycol                                                                     10.8                                                      lauryl phosphate    23.7                                                      oleic acid          26.2                                                                          100.0                                                     ______________________________________                                    

(total nitrogen, of the urea and valine, 8.07%)

Bacterial assay gave the number as 10² at the start and 10⁹ after 48hours. The rate of degradation of the petroleum was 84% by the sixthday.

Comparison with Example 25 shows that addition of the amino-acid allowed10⁹ bacteria to be obtained in place of 2.5×10⁸ with urea alone after 38hours. The degradation was of the same order (84% against 83%), but wasobtained more rapidly, in six days, whilst it was necessary to provide 7days in the case of urea alone.

EXAMPLE 27

According to the technique of Examples 25 and 26 a microemulsion wasutilized in which a part of the urea was replaced by an aqueous extractof maize cobs containing a series of amino-acids, predominantly alanine,arginine, glutamic acid and leucine. Other amino-acids present in lesserproportions were proline, isoleucine, threonine, valine, phenyl-alanine,methionine and cystine. The total nitrogen content in this extract was1%.

The microemulsion had the weight composition:

    ______________________________________                                        urea                 12.4%                                                    corn-cob extract    18.7                                                      butyl ether of ethyleneglycol                                                                     19.2                                                      lauryl phosphate    29.1                                                      oleic acid          20.6                                                      ______________________________________                                    

The water of the aqueous phase was that of the aqueous extract. Thetotal nitrogen content of the aqueous phase amounted to 6%.

Starting at 10² bacteria, they numbered 4×10⁹ after 48 hours and therate of degradation of the petroleum had attained 88% after six days.

Comparison of these results with those of Example 25 shows thedesirability of the addition of amino-acids to urea.

EXAMPLES 28 TO 37

In this series of tests analogous to Example 27, the lauryl phosphatewas replaced by oleyl phosphate and oleic acid by various liquidsindicated in the following table, which gives the rate of degradation ofpetroleum obtained after 6 days.

    ______________________________________                                        Example No                                                                              Hydrophobic liquid utilized                                                                      Degradation %                                    ______________________________________                                        28        Arachidic oil      88                                               29        Rape oil           82                                               30        Tall oil           86                                               31        Mixture of coconut fatty acids                                                                   85                                                         with 10% oil of vaseline                                            32        Lauric acid liquefied with                                                                       84                                                         10% of crude petroleum                                              33        Butyl caproate     85                                               34        Ethyl laurate      87                                               35        Methyl oleate      86                                               36        Amyl stearate      88                                               37        Gas oil with 10% sesame oil                                                                      83                                               ______________________________________                                    

Similar results are obtained with a mixture of C₁₂ -C₁₄ alkyl mono-, di-and tri(alkyltetraglycolether)-o-phosphates, known commercially underthe name "HOSTAPHAT KL 340 N", instead of oleyl phosphate.

We claim:
 1. A nutrient liquid composition intended to be added to ahydrophobic medium for the culture of microorganisms therein comprisinga microemulsion of an aqueous solution of nutrient material in a liquidimmiscible with water, the aqueous solution being the internal phase ofthe microemulsion, said microemulsion comprising by weight 10-30% ofwater, 4-10% of microorganism assimilable nitrogen in the form ofnitrogenous compounds, 5-35% of a 10-18 carbon atom alkyl or alkenylphosphate or an epoxylated phenol alkyl phosphate, 0-20% of an alkylether of an alkylene glycol and 20-50% of a fatty acid ester, acid oralcohol or mixture thereof with a petroleum derivative hydrocarbon. 2.Composition according to claim 1, wherein the nitrogenous compoundcomprises urea.
 3. Composition according to claim 2, wherein the urea ispresent in the aqueous solution at a concentration of 10-60% by weight.4. Composition according to claim 3, wherein said urea solution containsan amino acid.
 5. Composition according to claim 4, in which the ureanitrogen is 50-99% of the total nitrogen present.
 6. Compositionaccording to claim 5, in which the amino acid is at least one member ofthe group consisting of glycine, alamine, serine, cysteine, valine,glutamine, leucine, lysine, argynine, proline, tyrosine, aspartic acidand glutamic acid.
 7. Composition according to claim 5, in which saidmicroemulsion contains 20-50% of a mixture of a fatty ester, acid oralcohol with a petroleum derivative hydrocarbon in which the petroleumderivative hydrocarbon is 5-90% thereof.
 8. Composition according toclaim 1, wherein the nitrogenous compound is at least one memberselected from the group consisting of ammonium sulphate, ammoniumphosphate, lecithin and urea.
 9. Composition according to claim 8, inwhich at least two nitrogenous compounds are present, one of which isurea.
 10. Composition according to claim 1, in which said microemulsioncontains 20-50% of a mixture of a fatty ester, acid or alcohol with apetroleum derivative hydrocarbon in which the petroleum derivativehydrocarbon is 5-90% thereof.
 11. Composition according to claim 1, inwhich the external phase of the microemulsion is a lipomiscible organicliquid containing compounds of carbon assimilable by microorganisms. 12.Composition according to claim 1, in which the phosphorus to nitrogenweight ratio is from 0.02 to 0.2.
 13. Composition according to claim 12,wherein said ratio is from 0.05 to 0.15.
 14. Composition according toclaim 13, wherein said ratio is about 0.05.
 15. Composition according toclaim 1 containing 5-8% of assimilable nitrogen.
 16. Compositionaccording to claim 1 in which the fatty acid ester, acid or alcoholcomprises oleic acid.
 17. Composition according to claim 1 in which saidmicroemulsion comprises water, urea, oleic acid and oleyl phosphate. 18.Composition according to claim 1, wherein said microemulsion compriseswater, urea, oleic acid, butyl ether of ethylene glycol and laurylphosphate.
 19. Composition according to claim 1, wherein saidmicroemulsion comprises 13.8-19.2% water, 13.8-15.7% urea, 28-36.8%oleic acid, 10.5-10.6% butyl ether of ethylene glycol and 19.5-25% oflauryl phosphate.
 20. Composition according to claim 1 in which theinternal phase is droplets of 80-600 Angstroms in diameter.